Summary Obesity and the metabolic syndrome are prevalent in the United States and responsible for morbidity and mortality. The studies in this application aim to determine if a specific metabolic phenotype (increased reliance on anaerobic glycolysis in skeletal muscle and metabolic inflexibility) in overweight individuals is evident and approximates that of the severely obese. Overweight subjects will be selected for high and low fasting lactate concentrations (an indicator of anaerobic glycolysis) and undergo a glucose clamp with a muscle biopsy obtained in the resting state (Aim 1). A severely obese group will undergo the same tests with the primary variables being basal and insulin stimulated in vivo muscle substrate oxidation (near infrared spectrospopy; NIRS), muscle lactate release (microdialysis), RER, and muscle homogenate oxidation of pyruvate and fatty acids. Secondary variables are resting/fasting plasma lactate, whole body acetate oxidation, muscle fiber type, oxygen consumption in permeabilized fibers exposed to various substrates, quantities of mitochondrial proteins, VO2peak, REE, RER, body shape (3D-scanner) and body composition (DEXA). In Aim 2a, data from the clamp will be used to assess metabolic flexibility. The primary variables are metabolic flexibility (change in RER in response to insulin + glucose); substrate oxidation (NIRS and muscle homogenate oxidation of pyruvate and fatty acids); and anaerobic glycolysis (muscle lactate release during the clamp). Secondary variables are peripheral and hepatic insulin sensitivity. In Aim 2b, responses to a 3-day high fat diet will be determined and muscle biopsies obtained before and after the diet. The primary variables are indices of metabolic flexibility in response to the HFD and substrate oxidation (NIRS, muscle homogenate oxidation of pyruvate and fatty acids) and in vivo muscle lactate production. Secondary variables are peripheral and hepatic insulin sensitivity. Aim 2c will examine the ability of primary cell cultures derived from the research subject to respond to insulin or lipid incubation. The primary variables are metabolic flexibility in response to glucose + insulin (insulin stimulation of glucose oxidation and lactate production) and metabolic flexibility in response to fat (increase in fatty acid oxidation after 24 hr incubation with fatty acids). Aim 3 will determine if an intervention (gastric bypass surgery) which restores metabolic flexibility in severely obese individuals will also reduce dependence on anaerobic glycolysis. Subjects will undergo a glucose clamp and a muscle biopsy before and after the surgery. Primary variables are metabolic flexibility (change in RER in response to glucose + insulin during a clamp), substrate oxidation (NIRS, muscle homogenate oxidation of pyruvate and fatty acids) and in vivo muscle lactate release. Secondary variables are resting/fasting lactate, peripheral and hepatic insulin sensitivity, whole-body acetate oxidation, and metabolic flexibility of cultured muscle cells. The intent of this research is to determine indices of individuals at risk for the subsequent development of obesity.